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BOR - Papers in Press, published online ahead of print September 6, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.054056
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BIOLOGY OF REPRODUCTION 75, 816–822 (2006)
DOI: 10.1095/biolreprod.106.054056
© 2006 by the Society for the Study of Reproduction, Inc.

research-article

Effects of 25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 on Cytokine Production by Human Decidual Cells1

Katie N. Evans 3, Lisa Nguyen 4, Junny Chan 5, Barbara A. Innes 6, Judith N. Bulmer 6, Mark D. Kilby 5, and Martin Hewison 2 3 4

Division of Medical Sciences,3 Institute of Biomedical Research, The University of Birmingham, Birmingham, B15 2TT, United Kingdom Division of Endocrinology,4 Diabetes and Metabolism, Burns and Allen Research Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048 Department of Maternal and Fetal Medicine,5 Division of Reproduction and Child Health, Birmingham Women's Hospital, Birmingham, B15 2TH, United Kingdom School of Clinical and Laboratory Sciences (Pathology),6 University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United Kingdom

ABSTRACT

The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) is a potent immunomodulatory seco-steroid. We have demonstrated that several components of vitamin D metabolism and signaling are strongly expressed in human uterine decidua from first trimester pregnancies, suggesting that locally produced 1,25(OH)2D3 may exert immunosuppressive effects during early stages of gestation. To investigate this further, we used primary cultures of human decidual cells from first and third trimester pregnancies to demonstrate expression and activity of the enzyme that catalyzes synthesis of 1,25(OH)2D3, 1alpha-hydroxylase (CYP27B1). Synthesis of 1,25(OH)2D3 was higher in first trimester decidual cells (41 ± 11.8 fmoles/h/mg protein) than in third trimester cells (8 ± 4.4 fmoles/h/mg protein; P < 0.05). Purification of decidual cells followed by quantitative RT-PCR analysis showed that CYP27B1 was expressed by both CD10+VE stromal-enriched and CD10–VE stromal-depleted cells, with higher levels of mRNA in first trimester pregnancies. Expression of CYP27B1 correlated with TLR4 and IDO. Functional responses to 1,25(OH)2D3 were studied using CD56+ve natural killer (NK) cells isolated from first trimester decidua. Decidual NK cells treated with 1,25(OH)2D3 or precursor 25-hydroxyvitamin D3 (25OHD3) for 28 h showed decreased synthesis of cytokines, such as granulocyte-macrophage colony stimulating factor 2 (CSF2), tumor necrosis factor, and interleukin 6, but increased expression of mRNA for the antimicrobial peptide cathelicidin antimicrobial peptide. These data indicate that human decidual cells are able to synthesize active 1,25(OH)2D3, particularly in early gestation, and this may act in an autocrine/paracrine fashion to regulate both acquired and innate immune responses at the fetal-maternal interface.

1{alpha}-hydroxylase, cytokines, decidua, natural killer cell, placenta, pregnancy, steroid hormones, vitamin D

INTRODUCTION

The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), is a pluripotent seco-steroid, the putative biological actions of which extend far beyond its established effects on calcium homeostasis and bone metabolism [1–5]. In particular, 1,25(OH)2D3 has been shown to act as a potent modulator of both innate and acquired immune responses, including: enhanced macrophage bacterial killing [6, 7]; suppression of natural killer (NK) cell function [8]; inhibition of dendritic cell (DC) maturation [9–12]; inhibition of T-cell proliferation [12, 13]; and modulation of T-cell phenotype [12, 14, 15]. These observations have highlighted therapeutic applications for 1,25(OH)2D3 in the treatment of autoimmune disease and host-graft rejection [1–5], and have led to the development of synthetic analogs of vitamin D that retain the immunosuppressive, antiproliferative effects of 1,25(OH)2D3, while minimizing adverse calciotropic side effects [2, 16, 17]; however, vitamin D may also play a role in modulating normal immune responses. DCs and macrophages both express the enzyme 25-hydroxyvitamin D-1{alpha}-hydroxylase (CYP27B1) that catalyzes synthesis of 1,25(OH)2D3 from its inactive precursor, the major circulating form of vitamin D, 25-hydroxyvitamin D3 (25OHD3) [18–20]. This, coupled with the fact that many cells within the immune system express the nuclear receptor for 1,25(OH)2D3 (vitamin D receptor [VDR]) has supported the idea that vitamin D can act as an autocrine/paracrine regulator of immune responses [21–23].

A site of immune privilege that may act as an important target for vitamin D is the placenta—specifically, the maternal decidua—which forms the interface with newly implanted fetal placental trophoblast cells. The immunological nature of implantation is illustrated by the accumulation of subsets of cells of bone marrow origin (maternally recruited immune cells), including macrophages [24, 25], T-cells [26, 27], and a granulated lymphocyte population (termed uterine NK cells [uNK]) [28, 29] in decidual tissue during the first trimester of pregnancy. It has previously been hypothesized that 1,25(OH)2D3 may help to support successful implantation by attenuating decidual T-cell function [30]. In common with other sites of vitamin D-mediated immune regulation, the effects of 1,25(OH)2D3 in decidua appear to be due to autocrine/paracrine activation. The placenta and decidua were one of the first extrarenal sources of CYP27B1 activity to be documented [31, 32], and, more recently, we have defined the ontogeny of this enzyme during human pregnancy [33, 34]. Here we have used primary culture models to characterize the functional consequences of localized synthesis of 1,25(OH)2D3 by human decidual cells (defined as all cell types that are present within the decidua, including stromal cells, epithelial cells, lymphocytes, macrophages, and endothelial cells), particularly in the first trimester of pregnancy. Functional analyses indicate that 1,25(OH)2D3 is a potent regulator of cytokine production by uNK cells, suggesting a potential role for vitamin D in the immunoregulation of implantation.

MATERIALS AND METHODS

Tissue Samples

Tissue from first trimester decidua (gestational age between 10.3 ± 1.4 wk) was collected after surgical termination of apparently normal pregnancy for reasons unrelated to the study. Decidual tissues from third trimester pregnancies were collected at the time of elective caesarean section (performed due to previous surgery, breech birth, or at the mother's request) by blunt curettage of the uterine cavity at the site of placental insertion. All subjects gave informed written consent according to local ethics committee approval (Newcastle upon Tyne Hospitals and Birmingham Women's Hospital). Third trimester samples were 39.3 ± 1.8 wk gestational age. All tissues were obtained in accordance with the Polkinghorne Review of guidance on the research use of fetuses and fetal tissues.

Purification of Human Decidual Cells

Isolation of stromal and nonstromal enriched cell populations. Decidua was identified by its characteristic macroscopic appearance and histology and, after fine mincing under sterile conditions, was subjected to two vigorously shaken collagenase (used at a final concentration of 0.03 g/ml) (Sigma Chemical Co., Pool, U.K.) digestions of 30 min at room temperature. The resulting cell suspension was centrifuged at 300 x g for 1 min; the supernatant was then removed and sieved through a 40 µm cell strainer, followed by further centrifugation at 200 x g for 10 min. The remaining cell pellet was resuspended in RPMI 1640 media and cell counting performed. Immunomagnetic bead selection (Miltenyi Biotec, Surrey, U.K.) was used to isolate CD10-positive (CD10+VE) stromal-enriched cells. CD10 has been shown to be expressed by endometrial stromal cells, including decidualized stromal cells [35, 36]. Briefly, unfractionated decidual cells were incubated with primary mouse monoclonal anti-CD10 antibody (1/100 dilution; Novocastra Laboratories, Newcastle upon Tyne, U.K.) for 30 min at 4°C. The cells were then washed with ice-cold PBS and recentrifuged for 10 min at 200 x g. The resulting cell pellet was resuspended in Midi-Macs buffer (per manufacturer's instructions) and incubated with 2 µl Macs goat anti-mouse IgG micro beads/1 x 106 cells (Miltenyi Biotec) for 15 min at 4°C. This cell suspension was then passed through a Midi-Mac separation column (Miltenyi Biotec). A nonbound, CD10–VE stromal cell-depleted fraction was initially isolated. After two washes in Midi-Mac buffer, bound CD10+VE stromal-enriched cells were then eluted. In addition to the isolation of RNA, aliquots of each cell fraction were retained for cytospins to estimate selection efficiency by a modified immunohistochemical labeling technique [37] (80%–90% purity; data not shown).

Isolation of decidual uNK cells. Decidual cells from first trimester pregnancies were initially prepared by collagenase digestion, as described above. However, in this case, the cells were cultured in RPMI medium supplemented with 10% fetal calf serum (FCS) as a heterogeneous, unfractionated, mixed decidual cell suspension overnight (16 h). At this stage, the decidual cells were treated with 1,25(OH)2D3 (a kind gift from Dr. Lise Binderup, Leo Pharmaceuticals, Ballerup, Denmark), 25OHD3 (Sigma), or vehicle (0.1% ethanol) to ensure that all cells were exposed to the vitamin D metabolites. Positive selection of CD56+VE uNK cells was then carried out by immunomagnetic isolation of cells, as described above for CD10, using anti-CD56 antibody (Coulter, High Wycombe, U.K.). The resulting uNK cells were cultured for a further 12 h in RPMI with 10% FCS in the presence or absence of appropriate 1,25(OH)2D3, 25OHD3, or vehicle treatment. The resulting cells were used to carry out killing assays, while the remaining cell-free supernatants were analysed for cytokine expression, as outlined below.

RNA Extraction and Reverse Transcription

RNA was extracted from decidual stromal cell-enriched and depleted cell fractions with the GenElute Mammalian Total RNA kit (Sigma), as described previously [33], and DNase treated as indicated by the manufacturer (Sigma). The DNA-free RNA was then stored at –80°C. Aliquots (1.5 µg) of RNA from each DNase-treated sample were reverse transcribed using avian myeloblastosis virus reverse transcriptase, per the manufacturer's protocol (Promega, Southampton, U.K.), and samples were stored at –20°C.

Quantitative PCR Analysis of Gene Expression

Expression of specific mRNAs was quantified using an ABI 7700 sequence detection system (PE Biosystems, Warring, U.K.), as described previously [33]. Briefly, aliquots (25 µl) of PCR reactions were set up containing: TaqMan Universal PCR Master Mix in a 2x solution (PE Biosystems); 3 mmol/L Mn(Oac)2; 200 µmol/L dNTPs; 1.25 U Amplitaq Gold polymerase; 1.25 U AmpErase uricil-N-glycosylase; 5 or 1.25 pmol/µl TaqMan probe, 5 or 9 pmol/µl primers; and approximately 50 ng of cDNA were used per reaction. All reactions were multiplexed with the housekeeping gene 18S rRNA, provided as an optimized control probe labeled with VIC fluorochrome (PE Biosystems), enabling data to be expressed in relation to an internal reference to allow for differences in sampling. All fluorogenic probes for genes of interest were labeled with 5-carboxy fluorescein. Data were obtained as the cycle number at which logarithmic PCR plots cross a calculated threshold line (Ct values), according to the manufacturer's guidelines, and used to determine {Delta}Ct values (Ct of target gene – Ct of housekeeping gene, 18S rRNA). All reactions were performed in triplicate and expressed as a mean of these values. Samples were amplified with the following primers and probes: CYP27B1, forward primer 5'-TTGGCAAGCGCAGCTGTAT-3', reverse primer 5'-TGTGTTAGGATCTGGGCCAAA-3', TaqMan probe 5'-TTGCAATTCAAGCTCTGCCAGGCG-3'; VDR, forward primer 5'-CTTCAGGCGAAGCATGAAGC-3', reverse primer 5'-CCTTCATCATGCCGATGTCC-3', TaqMan probe 5'-AAGGCACTATTCACCTGCCCCTTCAA-3'; 24-hydroxylase (CYP24), forward primer, 5'-CAAACCGTGGAAGGCCTATC-3'; reverse primer 5'-AGTCTTCCCCTTCCAGGATCA-3'; TaqMan probe 5'-ACTACCGCAAAGAAGGCTACGGGCTG-3'; CD14, Assays-on-Demand (ABI) primer and probe mix Hs00169122_g1; Toll-like receptor 4 (TLR4), Hs00152939_m1; parathyroid hormone-related peptide (PTHLH), Hs00174969_m1; indoleamine 2,3-dioxygenase (IDO), Hs00158032_m1; interferon {gamma} (IFNG), Hs00174143_m1; cathelicidin antimicrobial peptide (CAMP), Hs00189038_m1; vascular endothelial growth factor (VEGF), Hs00900056_g1; placental growth factor (PLGF), Hs01123047_m1. Complimentary DNAs were amplified under the following conditions: 50°C for 2 min and 95°C for 10 min, followed by 44 cycles of 95°C for 15 sec and 60°C for 1 min.

Analysis of CYP27B1 Activity in Tissue Samples

Activity levels for CYP27B1 in primary cultures of first and third trimester decidual cells were assessed by quantifying the conversion of radiolabeled 25OHD3 to 1,25(OH)2D3 in homogenates from these tissues. For each assay, 10 nM [3H]-25OHD3 (specific activity, 152 Ci/mmol; Amersham, London, U.K.) was added to aliquots (200 µl) of PBS-washed collagenase-digested decidual cells resuspended in serum-free media to give 1 x 106 cells/assay. Cells and substrate were incubated for 5 h at 37°C, and the reaction terminated by freezing at –20°C. Vitamin D metabolites were then extracted from the reaction mixtures in 2.5 ml chloroform:methanol (4:1, vol:vol), the resulting organic phase dried under nitrogen, and extracts resuspended in 50 µl dichloromethane. The conversion of [3H]-25OHD3 to active [3H]-1,25(OH)2D3 was determined by scanning analysis of thin layer chromatography, as described previously [38]. Results were expressed as the mean femtomoles of 1,25(OH)2D3 produced per h per 106 cells.

Analysis of Cytokine Production

The impact of vitamin D on decidual cytokine expression was assessed using primary cultures of cells from first trimester decidua. Nonpurified whole populations of decidual cells were isolated from first trimester tissue and treated for 16 h with 1,25(OH)2D3 (100 nM) or 25OHD3 (100 nM) to facilitate potential activation of 25OHD3 to 1,25(OH)2D3 by either stromal or nonstromal cells. These cultures were then used for positive immunomagnetic selection of CD56+VE uNK cells, as previously described [37]. Using this technique, purity of CD56+ cells was consistently greater than 95%. Isolated CD56+VE uNK cells were then cultured for a further 12 h in the presence or absence of 1,25(OH)2D3 or 25OHD3 (both 100 nM). Cell-free supernatants from these cultures were then analyzed for 10 specific cytokines by using a multiplex assay system (Beadlyte Human Multi-Cytokine Detection System 3; Upstate Biotechnology, Lake Placid, NY), and cytokine levels were quantified using the Luminex 100 system (Luminex, Austin, TX), following the manufacturer's instructions. The concentrations of individual cytokines were determined from a standard curve assayed at the same time with defined cytokine reference samples using granulocyte-macrophage colony stimulating factor 2 (CSF2), IFNG, interleukin 4 (IL4), and tumor necrosis factor alpha (TNF).

Uterine NK Cell Chromium Release Assay

Decidual CD56+ non-MHC restricted effector cell function was assayed in a standard, 4-h chromium51 release assay against K562 target cells, as described previously [39]. The percentage specific chromium release (% SCR) from target cells was calculated from the equation:


Formula 75

75
In order to allow direct comparison between samples, the results were presented as the % SCR at an effector:target (E:T) ratio of 32:1 (%SCR32). This value was obtained using linear regression analysis (Microsoft Excel, Microsoft Corporation).

Data Analysis

Data from quantitative RT-PCR were reported as either the fold change ± SD or as the raw average {Delta}Ct values. Statistical analysis was performed on the triplicate average raw data using one-way ANOVA with Student-Newman-Keuls multiple comparison post-test (SPSS 11.5) and Pearson product moment correlation (SPSS 11.5). Data derived from the chromium release assay and cytokine analyses were analysed using the Student t-test (Microsoft Excel 2004).

RESULTS

Synthesis of 1,25(OH)2D3 in Decidua from First and Third Trimester Pregnancies

In previous studies we have reported increased expression of mRNA and protein for CYP27B1 in first trimester biopsies of human decidua when compared to third trimester biopsies [39]. To assess the relationship between this and 1,25(OH)2D3 production we used primary cultures of nonselected human decidual cells from first and third trimester pregnancies to quantify metabolism of radiolabelled 25OHD3. Data (Fig. 1) confirmed that CYP27B1 activity was higher in first trimester decidual cells (41 ± 11.8 fmoles/h/106 cells) compared to third trimester cells (8 ± 4.4 fmoles/h/106 cells) (P < 0.05). In contrast to CYP27B1, CYP24 activity (production of 24,25[OH]2D3 from 25OHD3) was not detectable in either first or third trimester decidua (data not shown).


Figure 75060901
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  		FIG. 1. Synthesis of 1,25(OH)2D3 in cultures of human decidual cells from first and third trimester (1st and 3rd T) pregnancies. Asterisk indicates statistically different from third trimester decidua, P < 0.05.

Expression of CYP27B1, VDR, and CYP24 in Decidual Cell Subpopulations

To determine whether the higher levels of decidual CYP27B1 expression and activity in early gestation were associated with a specific cell subpopulations, we carried out further studies using stromal cell-enriched (CD10+VE) and stromal cell-depleted (CD10–VE) populations of cells from first and third trimester decidua. The stromal cells comprised decidualised endometrial stromal cells showing the characteristic morphological features of this cell type. Data in Figure 2 indicated that the pattern of expression for CYP27B1 mRNA in primary decidual cells was similar to that previously described for decidual biopsies with increased levels in first trimester compared to third trimester. In first trimester, expression of CYP27B1 was highest in CD10+VE stromal-enriched cells (P < 0.05) but the opposite was true in third trimester cells (P < 0.001). The high level of CYP27B1 in CD10+VE cells in first trimester decidua was accompanied by elevated expression of CYP24 when compared to first trimester CD10–VE cells (P < 0.01), but there was no significant difference in the expression of this gene across gestation. Likewise, VDR expression was unaffected by either CD10 fractionation or gestation period.


Figure 75060902
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  		FIG. 2. Quantitative RT-PCR analysis of the expression of genes associated with vitamin D metabolism and signaling in stromal-enriched (CD10+VE) and stromal-depleted (CD10–VE) cells from first and third trimester (1st and 3rd T) human decidua. Data for CYP27B1, VDR, and CYP24 are shown as fold-change in gene expression relative to expression in CD10+VE third trimester cells as an arbitrary value of 1. Data are also shown as unmanipulated {Delta}Ct values (±SD), which were used to carry out statistical analyses. Higher {Delta}Ct values correspond to lower levels of mRNA expression. Asterisks indicate statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001.

Expression of CYP27B1 by CD10+VE and CD10–VE cells was also compared to potential regulators of 1,25(OH)2D3 production, including PTHLH, CD14 and TLR4; the latter two being components of the endotoxin receptor. CD14 and TLR4 are abundantly expressed on immune cells such as macrophages and initiate immune responses to bacterial cell wall components such as lipopolysaccharide (LPS). Trends of correlation with CYP27B1 expression were observed for all three gene products but a statistically significant (P < 0.01) correlation was only observed with TLR4 and CYP27B1 in stromal cell depleted CD10–VE decidual cells (Fig. 3). No correlation between CYP27B1 and PTHLH, TLR4, or CD14 was observed for third trimester decidual cells (data not shown).


Figure 75060903
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  		FIG. 3. Relationship between expression of CYP27B1, PTHLH, CD14, and TLR4 in stromal-enriched CD10+VE and stromal-depleted CD10–VE cells from first trimester decidua. Data show correlations between raw {Delta}Ct values with corresponding correlation coefficients (R) and P values. Higher {Delta}Ct values correspond to lower levels of mRNA expression.

Regulation of Decidual Cell Function and Cytokine Expression by 25OHD3 and 1,25(OH)2D3

The impact of vitamin D metabolites on decidual cytokine expression and uNK activity was assessed using primary cultures of cells from first trimester decidua. Data in Figure 4 show the changes in levels for each cytokine following culture with 1,25(OH)2D3 or 25OHD3, relative to vehicle-treated cells. The vitamin D metabolites potently suppressed cytokine expression with CSF2, IL1 and IL6, and TNF, showing statistically significant regulation by both 1,25(OH)2D3 and 25OHD3. By contrast, 1,25(OH)2D3 and 25OHD3 appeared to have no effect on uNK killing activity as determined by analysis of chromium release from CD56+VE cells. At an E:T of 32:1, cytotoxicity of CD56+VE uNK cells against K562 cells (mean ± SEM; n = 10) was 23.25 ± 4.91% in control samples with no added vitamin D metabolite, 20.75 ± 3.82% in the presence of 1,25(OH)2D3, and 19.65 ± 4.14% in the presence of 25OHD3.


Figure 75060904
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  		FIG. 4. Effect of 1,25(OH)2D3 and 25OHD3 on cytokine expression by CD56+VE uNK cells from first trimester decidua. Cells were treated for 28 h with 100 nM 1,25(OH)2D3 or 100 nM 25OHD3. Levels of the following cytokines: CSF2, IFNG, IL10, IL12, IL1B, IL2, IL4, IL6, IL8, and TNF were analyzed and data shown as percent change in expression relative to vehicle-treated cells ± SEM (n = 4 separate decidua). Asterisks indicate statistically different from vehicle-treated controls; *P < 0.05, **P < 0.01, *** P < 0.001. Values in parentheses show the mean values for absolute levels (pg/ml) of each cytokine in control (untreated) cultures of cells.

Another potential marker of immune responses in the decidua is the enzyme IDO, which has been proposed to play a role in fetal-maternal tolerance [40]. In a similar fashion to CYP27B1, mRNA levels for IDO were much higher in first trimester decidua relative to their third trimester equivalents (425-fold and 490-fold higher for stromal-depleted CD10–VE and stromal-enriched CD10+VE cells, respectively, both P < 0.001). Further analysis of mRNA expression in decidual cells showed that IDO expression correlated with CYP27B1 in both stromal-enriched CD10+VE and stromal-depleted CD10–VE cells from first trimester pregnancies (P < 0.05) (Fig. 5). The relationship between IDO and CYP27B1 expression in first trimester decidual cells appeared to be specific, as no similar correlation was observed for other mRNAs, such as VDR, CYP24, PTHLH, CD14, or TLR4 (data not shown). Furthermore, there was no correlation in expression between IDO and CYP27B1 in third trimester cells, indicating that the link between these two gene products was closest in early gestation. However, decidual cells treated with either 1,25(OH)2D3 and 25OHD3 (both 100 nM for 24 h) showed no change in mRNA for IDO, suggesting that the association between IDO and CYP27B1 was not due to synthesis of 1,25(OH)2D3 (Fig. 6). Interestingly, expression of mRNA for the T helper cell type 1 cytokine, IFNG, was decreased by either 1,25(OH)2D3 or 25OHD3, whereas expression of antimicrobial CAMP was increased by both metabolites. By contrast, expression of VEGF and PLGF unaffected by treatment with either 25OHD3 or 1,25(OH)2D3.


Figure 75060905
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  		FIG. 5. Relationship between expression of CYP27B1 and IDO in stromal-enriched CD10+VE and stromal-depleted CD10–VE cells from first and third trimester decidua. Data are shown as correlations between raw {Delta}Ct values with corresponding correlation coefficients (R) and P values. Higher {Delta}Ct values correspond to lower levels of mRNA expression.


Figure 75060906
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  		FIG. 6. Effect of 25OHD3 and 1,25(OH)2D3 on gene expression in primary cultures of human first trimester decidual cells. Nonpurified decidual cells from first trimester tissue biopsies (n = 5) were incubated with 25OHD3, 1,25(OH)2D3 (both 100 nM), or vehicle (ethanol) for 24 h and then used to extract RNA for RT-PCR analysis of: IFNG, IDO, CAMP, VEGF, or PLGF. Data shown are the fold-changes in expression of these genes in 25OHD3- or 1,25(OH)2D3-treated cells relative to vehicle-treated control cells. Values reported are the mean ± SD. Asterisks indicate statistically different from vehicle-treated controls; *P < 0.05, **P < 0.01.

DISCUSSION

The potential importance of vitamin D during pregnancy has been recognized for many years. Although concentrations of 1,25(OH)2D3 in the fetus are low, circulating maternal levels are strikingly increased during pregnancy before returning to normal after parturition [41]. This increase appears to be due primarily to classical renal synthesis of 1,25(OH)2D3 by the mother, but there is also evidence of CYP27B1 activity in the placenta/decidua, suggesting that these tissues also contribute to 1,25(OH)2D3 levels [31, 32]. Crucially, analysis of knockout mouse models for both the VDR and CYP27B1 genes has shown that the vitamin D pathway is not only important for fetal skeletal development, but may also be involved in normal reproductive function [42, 43]. In previous studies of human pregnancies we have shown that expression of the vitamin D-activating enzyme, CYP27B1, is most abundant in decidual tissue and is greatest in early stages of gestation [33]. Based on these observations and the established immunomodulatory properties of 1,25(OH)2D3, we have postulated that vitamin D may play an important role in human pregnancy by acting as an autocrine/paracrine regulator of immunity at the fetal-maternal interface [34]. Data presented here support this hypothesis by showing that both active 1,25(OH)2D3 and its inactive precursor, 25OHD3, can modulate decidual immune cell function. Initial studies using heterogenous as well as stromal-enriched and depleted decidual cell cultures highlighted several important facets of vitamin D metabolism and function in the decidua. First, as previously described, expression of CYP27B1 is much higher in first trimester pregnancies than third trimester pregnancies [33]. Changes in the level of expression for this enzyme are much greater than those observed for other vitamin D-related gene products, such as VDR or CYP24, re-emphasizing the potential importance of CYP27B1 during the early stages of gestation. In keeping with previous immunohistochemical analyses [33], expression of CYP27B1 did not appear to be restricted to one specific cell type, but was common to stromal and nonstromal cells. Collectively, these data indicate that it is possible to recapitulate ex vivo variations in decidual vitamin D metabolism using an in vitro model. This, in turn, allowed us to carry out subsequent studies aimed at defining the functional impact of vitamin D metabolites on decidual cell immunology.

The link between CYP27B1 and immune function in the decidua was further underlined by correlation with expression of TLR4. This endotoxin recognition factor was expressed at similar levels in first trimester stromal-enriched CD10+VE and stromal-depleted CD10–VE cells (data not shown), but significant correlation with CYP27B1 expression was only observed in the CD10–VE stromal-depleted decidual cells. This may reflect the high proportion of immune cells that make up the nonstromal cell population of decidua, including macrophages and DCs, both of which express TLR4 [44]. Relatively little is known about the expression and function of TLRs in fetal-maternal immunology. TLR4 and associated signaling peptides have been shown to be functionally active in human endometrium [45], and other studies have suggested a role for the endotoxin sensor in mediating bacterially induced preterm labor [46]. The principal ligand for TLR4, LPS, can act as a potent stimulator of CYP27B1 expression in macrophages [47], epithelial cells [38], and endothelial cells [48]. It therefore seems likely that a similar mechanism for induction of 1,25(OH)2D3 synthesis is present in human decidual cells, although the specific stimulus for TLR4 and the target cells expressing this receptor have yet to be determined.

To assess the significance of localized synthesis of 1,25(OH)2D3 with respect to decidual immune function, we investigated changes in uNK cell activity. Uterine NK cells constitute the predominant endometrial leukocyte population during early pregnancy and have, therefore, been widely studied with respect to their role in the innate immune responses associated with implantation and the potential importance of this in recurrent pregnancy loss [28]. When compared to NK cells from peripheral blood, uNK cells show some clear functional and phenotypic differences. In particular, they have relatively attenuated killing function, as determined by lytic activity against NK-sensitive targets [49]. This may explain, in part, the lack of response to added 25OHD3 or 1,25(OH)2D3 with respect to uNK cell killing activity. We did, however, observe potent changes in the uNK cell cytokine profile after treatment with either 1,25(OH)2D3 or 25OHD3, which was consistent with previous studies of peripheral blood NK cells [8]. Variable patterns of cytokine production have been reported previously for peripheral blood-derived CD56+VE cells reflecting the different subsets of CD56+VE cells, such as CD56dim (cytotoxic) and CD56bright [50, 51]. The picture is less clear for uNK. Transcripts for CSF2, TNF, IFNG, transforming growth factor (TGFB2), and leukemia inhibitor factor have been detected in purified decidual lymphocyte preparations [52]—and IL10 is also produced by uNK cells [53]. Data presented in this study suggest a broad cytokine profile for uNK cells in first trimester decidua, although some cytokines, such as IL10, IL12, and IL4, were less abundant than others. Regardless of absolute levels, the vitamin D metabolites consistently inhibited cytokine expression, underlining their potential role as modulators of uNK cell activity, particularly in early gestation. Cytokines, such as CSF2, IFNG, and TNF, have been suggested to play an important role in etiology of spontaneous early pregnancy loss [54]. It is therefore interesting to speculate that 1,25(OH)2D3 may help to support successful implantation by acting as a locally synthesized immunoregulatory hormone.

In this study, we have focused on the impact of vitamin D on uNK cells because of their relative abundance in first trimester decidua. However, 1,25(OH)2D3 is also known to regulate other facets of immunity, including the induction of innate immune responses [6, 7]. Thus, it is interesting to note that expression of mRNA for CAMP, an antimicrobial peptide, was enhanced following treatment of decidual cells with 1,25(OH)2D3 or 25OHD3. To our knowledge, this is the first report of CAMP expression by decidual cells, and the precise function of this innate immunity peptide within the decidua remains unclear. Decidual macrophages are known to be capable of initiating antibacterial responses to counteract intrauterine infection, a potential factor in preterm pregnancies [55]. However, many other cells are known to synthesize CAMP, including NK cells [56], and the peptide has been shown to exhibit diverse properties, such as effects on lymphocyte chemotaxis [56]. The fact that the gene for CAMP has a well characterized vitamin D response element in its promoter [57] suggests that this may be an important target for locally produced 1,25(OH)2D3 in early gestation, and further underlines the potential importance of vitamin D as a novel regulator of reproductive immunology.

FOOTNOTES

1Supported by BBSRC research grant no. BBS/B/01014. Back

Correspondence: 2 Martin Hewison, Division of Endocrinology, Diabetes, and Metabolism, Rm. D-3088, Burns and Allen Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048. FAX: 310 423 4550; e-mail: m.hewison{at}cshs.org

Received: 22 May 2006.

First decision: 26 June 2006.

Accepted: 28 August 2006.

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